1. Field of the Invention
Fluorescent compounds find a wide variety of applications. They find use in fluorescent immunoassays, histochemical staining, displays, inks, and the like. Of particular interest for the subject invention is the use of antigenic conjugates (includes receptor conjugates) with fluorescent compounds to be used in the determination of a variety of ligands, both antigens and receptors. A substantial proportion of the ligands are assayed in physiological fluids, such as serum, where the serum can provide substantial background fluorescence. One way to diminish the background fluorescence resulting from naturally present fluorescers is to provide a fluorescent compound which absorbs at relatively long wavelengths. The compound should desirably have a large Stokes shift, be stable under conditions of the assay, be relatively free of non-specific interference, both from materials in solution and the compound to which the fluorescer is conjugated, and provide high quantum yields. In addition, for certain applications, it is desirable that the fluorescer be coupled with a quencher molecule, that is a molecule which is capable of absorbing the energy of the fluorescer in the excited state, when within a predetermined distance, so that the fluorescer does not fluoresce.
2. Description of the Prior Art
A large number of fluorescein derivatives have been reported in the literature. The following are believed to be the most exemplary in relation to the subject invention and are reported in conjunction with the Chemical Abstracts citation. The numbering is based on the parent molecule 3',6'-dihydroxyspiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one.
Asymmetrical fluorescein derivatives without functionalities for attaching to other molecules have been reported in the literature. The following is not intended to be exhaustive of such compounds. The Chemical Abstracts reference is indicated. 2',4'-dichloro-7'-bromo, 2',7'-dichloro-4'-bromo, 2,7'-dibromo-4'-chloro, 2'-bromo-4',7'-dichloro, 2',4'-dibromo-7'-chloro, C.A. 62, 13116C; 2',7'-dibromo-4'-hydroxy, C.A. 61, 7407d; 2',4',7'-tribromo, C.A. 53, 9573h, 65, 4328a; 2',4',7'-trichloro, C.A. 55, 12393f, 63, 6954f; 2',4',5'-triiodo, C.A. 57, 11794b; 2',4',5'-tribromo, C.A. 53, 9573h; 2',4',5'-trichloro, C.A. 55, 12393f, 63, 6954f; 4',5'-dibromo-2'-iodo, C.A. 49, 254e; 4',5'-dinitro-2'-hydroxymercuri, C.A. 51, 313i; 2',5'-dichloro, 2',4'-dichloro, 4'-chloro, 2' -chloro, C.A. 55, 12393f, 63 6954f; 2'-bromo-5'-chloro, C.A. 62, 13116c; 2',5'-dibromo, C.A. 53, 9573g, 71, 8201e; 4'-[(4-sulfamoyl-O-tolyl)azo], 4'-[(p-[2-thiazolylsulfamoyl]phenyl)azo], 4'-[(p-sulfamoylphenyl)azo], 4'-[(6-sulfamoyl-m-tolyl)azo], 4'-[(p-[2-pyridylsulfamoyl]phenyl)azo], C.A. 72, 91456d; 4'-iodo, C.A. 57, 11794c; 4'-chloro, C.A. 63, 6954f, 70, 2018n; 4'-bromo, C.A. 62, 5249b, 71, 8201c; 2'-pentenyl, 2'-allyl, C.A. 31, 1388; 2'-benzyl, C.A. 74, 45540v; 2'-bromo, C.A. 53, 9573h, 62, 5249c, 71, 8201c.
Symmetrical fluorescein derivatives, having alkyl or oxy substituents may be found in co-pending applications Ser. No. 73,158, filed Sept. 7, 1979 and 73,163, filed Sept. 7, 1979, now U.S. Pat. Nos. 4,318,846 and 4,351,760, respectively.